Infections due to the fungus Cryptococcus neoformans have emerged as a major cause of morbidity and mortality in patients with impaired cell- mediated immunity (CMI), especially those with AIDS. This proposal focuses on defining specific and unique aspects of how lymphocytes contribute to host defenses against cryptococcosis. specific aims include: (1) Defining the lymphocyte populations in human blood capable of inhibiting growth of C. neoformans. Preliminary data demonstrate that freshly isolated and Interleukin (IL)-2-activated human NK cells and T cells are capable of directly attaching to an inhibiting the growth of C. neoformans. Important aspects of this interaction between lymphocytes and C. neoformans will be defined, including: (i) the exact phenotypes of the lymphocytes involved; (ii) the opsonic requirements; (iii) the ability of cytokines other than IL-2 to activate lymphocyte-mediated fungistasis; and (iv) the mechanism(s) by which lymphocytes mediate fungistasis. Both freshly isolated an cloned lymphocyte populations will be used. (2) Characterizing the nature of lymphocyte-C. neoformans intracellular conjugates. Preliminary data demonstrate both NK cells and T cells readily form conjugates with C. neoformans. Flow cytometry, videomicroscopy, and confocal scanning laser microscopy will be used to characterize the conditions under which conjugate formation occurs, the phenotypes of the lymphocytes forming conjugates, and the lymphocytic receptor(s) and fungal ligand(s) responsible for this interaction. (3) Investigating lymphocyte activation, cytokine gene expression and release following cryptococcal stimulation. Using fluorescent digital imaging microscopy and flow cytometry, intracellular calcium fluxes in individual lymphocytes will be determined as the cells attach to and spread over C. neoformans. Lymphocyte degranulation in response to cryptococcal stimulation will be determined by microscopic observation under Nomarski optics, and by measuring the release of BLT-esterase. Cytokine (IL-2, IL-4, interferon-gamma) gene expression and release by lymphocytes following direct and indirect cryptococcal stimulation will be determined. (4) Defining the conditions under which lymphoproliferation occurs in response to C. neoformans. Preliminary data demonstrate that naive donors have a weak lymphoproliferative response to whole C. neoformans unless the organisms are opsonized, and fail to respond to all to a panel of purified cryptococcal antigens. the specific opsonic requirements for lymphoproliferation will be determined in both naive subjects and patients who have recovered from cryptococcosis. In addition, the ability of patients to respond to purified cryptococcal antigens will be defined. Cytokine gene expression and release during the course of the lymphoproliferative response will be determined. Completion of these studies over the next five years should contribute important new information to our basic understanding of the immunology of cryptococcosis and may provide a rational basis for novel preventive and immunotherapeutic approaches to combating cryptococcosis.